Nitrocellulose membrane-poly (vinyl alcohol)-ionic imprinting (NCM-PVA-I-I) was prepared using Cu 2+ as template. The cavity in NCM-PVA-I-I matched Cu 2+ very well and the selectivity was high. Cu 2+ entered the cavity and then could form ionic association ([Cu 2+]·[(Fin −) 2]) with the anion of fluorescein (Fin −) outside the cavity by electrostatic effect. [Cu 2+]·[(Fin −) 2] could emit strong and stable room temperature phosphorescence on NCM-PVA-I-I. Its Δ I p was proportional to the content of Cu 2+. Based on the above facts, a new method for the determination of trace copper by solid substrate-room temperature phosphorimetry (NCM-PVA-I-I-SS-RTP, SS-RTP is the abbreviation of solid substrate-room temperature phosphorimetry) using NCM-PVA-I-I technique has been established. The linear range of this method was 2.00–144.00 fg Cu 2+ spot −1 (sample volume: 0.40 μL spot −1, corresponding concentration: 5.00–360.00 pg mL −1), and the detection limit calculated by 3Sb/ k was 0.43 fg Cu 2+ spot −1 (corresponding concentration: 1.1 × 10 −12 g mL −1, n = 11). Samples containing 2.00 and 144.00 fg Cu 2+spot −1 were measured, respectively, for seven times and R.S.D.s were 3.5% and 4.7%. NCM-PVA-I-I-SS-RTP could combine very well the characteristics of both the high sensitivity of SS-RTP and the high match and selectivity of NCM-PVA-I-I, and it was rapid, accurate, sensitive and with good repeatability. It has been successfully applied to determine trace copper in human hair and tea samples.